Cytokines of the tumor necrosis factor (TNF) family provide a unique point of view of biological regulation. These cytokines and their receptors are expressed in almost all cells and trigger a wide range of different, in part contrasting, cellular activities (Wallach et al., 1999, Locksley et al., 2001, and Wallach et al. 2000). They help to regulate practically every aspect of immune defense, as well as certain developmental processes. All of these activities are mediated by a unique set of apparently few signaling proteins, which are shared by the different receptors (Wallach et al., 1999).
Cytokines normally serve to enhance defense. However, when acting in excess, they may cause great damage, comparable to that which pathogens can cause. In fact, in many diseases, unwarranted effects of cytokines constitute a major pathogenic cause.
Cytokines of the TNF family regulate a wide range of different immune defense mechanisms, both of the innate and the adaptive types. Excessive function of several of them, including TNF, the Fas ligand, CD40 ligand and others have been implicated in the pathology of various diseases. There is, in particular, extensive evidence for a major pathological role of TNF in a wide range of diseases: infectious diseases such as malaria and sepsis, autoimmune diseases such as rheumatoid arthritis, inflammatory bowel diseases and psoriasis, and certain types of cancer. Indeed, blocking TNF action by agents such as anti-TNF antibodies or soluble TNF receptors was found to provide therapy at such situations (Beutler, 1999, Kollias et al., 1999 and Reimold, 2003). In some pathological situations, including rheumatoid arthritis and Crohn's disease, a rather significant proportion of the patients respond favorably to anti-TNF therapy. There are, however, also patients with such diseases that respond rather poorly to these agents, raising the need to find additional approaches for therapy (Andreakos et al., 2002).
Unlike many other cytokines that act solely as soluble proteins following their secretion by the cytokine-producing cell, the ligands of the TNF family are produced as cell-bound type II transmembrane proteins (with the exception of lymphotoxin which is produced as a soluble secreted protein). They can exert their effects in this form, affecting only cells that are located adjacently to the ligand-producing cell (juxtacrine regulation). Most of them are also shed, forming soluble molecules that circulate. Part of those soluble ligands, for example TNF, are capable of acting as soluble cytokines, serving as paracrine regulators (affecting cells located relatively close to the ligand producing-cells) and endocrine regulators (affecting remote cells). Some ligands of the TNF family, for example the Fas ligand, do not act effectively in their shed form and may in that form even serve as antagonist to the cell-bound form (Wallach et al., 1999 and Locksley et al., 2001).
The occurrence of ligands of the TNF family on the surface of the cells producing them provides a potential means for specific targeting of these ligand-producing cells. Such means can allow selective killing of the ligand-producing cells at situations where the ligand plays a pathogenic role.
In several respects, destruction of cells producing a cytokine may turn to provide even better defense against the pathogenic effects of this cytokine than just direct blocking of the function of the cytokine molecules. Destruction of the cytokine-producing cell prevents further synthesis of the cytokines and thus is likely to provide more durable protection than that obtained by just blocking the effect of the cytokine molecules that had been already synthesized.
Cells producing a cytokine often produce simultaneously some other cytokines that together serve to elicit a particular type of immune response. Well-known examples are the Th1- and Th2-type T lymphocytes, that produce distinct groups of cytokines, each serving to elicit a different type of immune defense (Jankovic et al., 2001). Destroying cells producing a cytokine may thus, besides arrest of the synthesis of that particular cytokine, also result in arrest of synthesis of several other cytokines that assist the former in its pathogenic effects.
While blocking circulating cytokines affects the whole body, killing cytokine-producing cells or attenuation of cytokine production can be restricted to a particular site in the body where these cells reside, thus allowing abolition of the cytokine deleterious effects at that particular site, while maintaining beneficial effects of the cytokine at other sites.
Studies of the effect of anti-TNF therapy in Crohn's disease suggest that killing of TNF-producing cells may, in some pathological situations, indeed provide more effective therapy than that obtained by just blocking TNF. Therapeutic effects of anti-TNF antibodies in this disease were found to correlate with early induction of death of the TNF-producing cells by the antibodies (Lugering et al., 2001, Van Deventer, 2001, and Van den Brande et al., 2003).
TNF-α is expressed by activated monocytes, macrophages and CD8+T cells. These cells present membrane-anchored TNF-α as a key component in their cytolytic pathway. In the following diseases activated macrophages are suspected to be involved in their pathology: septic shock, rheumatoid arthritis, ankylosing spondylitis and psoriatic arthritis (Singh and Suruchi, 2004), psoriasis (Asadullah et al., 2002), amyotrophic lateral sclerosis (Ghezzi et al., 1998), insulin-dependent diabetes mellitus (Kagi et al., 1999), graft-versus-host disease (Hongo et al., 2004), and sickle cell anemia (Belcher et al., 2000). In the following diseases activated CD8+T cells are suspected to be involved in their pathology: systemic lupus erythematosus (Pacheco et al., 2002), reactive arthritis and other autoimmune diseases (Mittler, 2004). Sometimes, activated CD8+T is detrimental to a specific organ. For example, extensive hepatocyte apoptosis which occurs during liver inflammation, is induced following infiltration of activated CD8+T cells to the liver.
Chimeric molecules comprised of a cytotoxin linked to a targeting molecule that binds to a cell-surface constituent can serve as potent cell-killing agents. Choosing a targeting moiety that recognizes a cell-type specific surface constituent can allow applying such cytotoxic chimera for selective destruction of specific cells in vivo. For example, chimeric fusion proteins comprised of antibodies against cancer-specific epitopes fused to Pseudomonas exotoxin (PE) or to Diphtheria toxin (DT) can specifically target and kill cancer cells. Such anti-cancer effects have also been obtained with chimera in which the toxins have been fused to hormones or ligands such as IL-2, IL-4 or IL-13, whose receptors are prevalent in certain tumors. Likewise, cytotoxin-containing chimera were designed to be targeted to pathogen-afflicted cells. For example, HIV-infected cells can be selectively destroyed using immunotoxins comprised of an anti-gp120 antibody directed to the conserved CD4 binding site of gp120, or CD4, attached to a Pseudomonas exotoxin (Brinkmann and Pastan, 1995, Pastan and Kreitman, 1998 and Pastan, 2003).
One kind of possible agents for targeting cytotoxins or other modulating agents to cells that express ligands of the TNF family are antibodies against these ligands. Indeed, antibodies against the CD40 ligand have been applied to target a toxin to CD40 ligand producing cells (EP 1005372).
U.S. Provisional Application No. 60/582,827 discloses a chimeric protein (TBP-PE38) comprising the extracellular portion of the p55 TNF receptor (TBP-1) attached to the Pseudomonas aeruginosa exotoxin translocating and ADP-rybosilating domains (PE38 or PE I-II). It was shown that TBP-PE38 binds epithelial cells overexpressing surface TNF and induces cytotoxicity in such cells.
A variety of bacterial toxins specifically bind to receptors on the cell surface and are internalized via receptor-mediated endocytosis. In general, receptor-mediated endocytosis allows the selective up-take of extracellular proteins (e.g. receptors) and small particles (e.g. ligands) (Molecular cell biology Darnell Lodish and Baltimore). Typically, after binding of a particle to a receptor on the plasma membrane, the receptor-ligand complex is internalized in a clathrin-coated pit that pinches off to become a clathrin-coated vesicle. Then, the clathrin coat depolymerizes to triskelions, resulting in uncoated vesicle, often called endosome. The endosome fuses with an uncoupling vesicle called CURL (compartment uncoupling receptors and ligands), that is characterized by internal pH of about 5.0. The low pH causes the particles to dissociate from the receptor. A receptor-rich region buds off to form a separate vesicle that recycles the receptor back to the plasma membrane.
Proteins internalized by receptor-mediated endocytosis undergo various fates. For example, they can be transferred to the lysosomes, where they are destroyed, they may be minimally processed and remain in the cells, or in other cases the endocytosed material may pass all the way through the cell membranes and exocytosed, or secreted from the plasma membrane at the opposite site.
Pseudomonas exotoxin A (PE) binds and enters cells via a α2-macroglobulin receptor/low density lipoprotein receptor-related protein. Following internalization, PE is proteolitically processed in the endosome by a furin-like protease, reduced to a 38 kDa active fragment (PE-38) and such fragment is translocated to the cytosol where it ADP ribosylates elongation factor 2 causing protein synthesis inhibition.
Both proteolytic processing of PE (carried out in the endosome or in the endoplasmic reticulum by furin-like proteases) and translocation of PE-38 to the cytosol are necessary for effective cell toxicity. Domain I of PE (FIG. 1) includes the cell-binding activity, domain II the translocating activity, and domain III the ADP-rybosylating activity. Each one of these domains is comprised of structural units which can be independently refolded. The translocating domain (PEII) contains six consecutive a-helices (A-F). Deletion mapping studies based on the primary structure of the protein showed that the amino-terminal segment encompassing the A and B helices are required for PE translocation (Siegall et al., 1989). At the carboxyl-terminal end, some residues within the E helix were found to be critical, whereas other residues and the entire F helix could be deleted without apparent loss of translocation activity. Moreover, deletion of the last α-helix (F) both enhanced translocation activity and cytotoxicity of PE (Taupiac et al., 1999).
Although there is evidence that the portion of PE exotoxin including PE I-II is capable of translocating barnase, a bacterial enzyme, from the endosome to the cytoplasm of eukaryotic cells (Prior et al., 1996), the translocating activity of domain II flanked by heterologous proteins was not reported nor demonstrated.
Pseudomonas exotoxin and diphtheria toxin are powerful cytotoxic agents. Pseudomonas exotoxin and diphtheria toxin inhibit protein synthesis affecting the entire cellular metabolism in a wide variety of cells. In contrast, other type of bacterial toxins are known which regulate specific metabolic pathways. For example, each one of the six Yersinia's outer protein (Yop) toxins modulates specific signaling pathways.
Yersinia pestis is the causative agent of bubonic plague. Yersinia pestis is responsible for three human pandemics: the Justinian plague (VI-VIII centuries), the Black Death and the modern plague. Yersinia enterocolitica and Yersinia pseudotuberculosis are food-born pathogens, which cause gastroenteritis. The pathogenicity of Yersinia results from its ability to overcome the immune defense of the mammalian host.
Yersinia's outer protein P (YopP) is a toxin produced by Y. enterocolitica. YopP is essential for the establishment of systemic infection in mice and inhibits production of TNF-α and triggers apoptosis in infected macrophages (Cornelis, Nature 2001). Cytosolic delivery of YopP, which involves translocation across the bacterial double membrane envelope and the host cell membrane, is triggered by an activated protein secretion machinery (type III protein secretion system), which is activated upon host-cell contact. Such protein secretion machinery is absolutely needed for delivery of YopP to the cytosol.
YopP is thought to activate apoptosis through one of two routes, either by activating of BID/caspase 8 pathway and preventing the release of anti-apoptotic factors, or through a less ‘direct’ pathway involving inhibition of NF-kB. YopP prevents phosphorylation of the NF-kB inhibitor by IKK, and therefore inhibits migration of NF-kB to the nucleus. YopP also causes inhibition of the mitogen-activated protein kinase (MAPK) signaling pathway by inhibiting the upstream MAPK kinases (MEKs). As a result of these inhibitory actions of YopP, transcription activators such as the cAMP-response-element-binding protein (CREB) and activating transcription factor (ATF)-1, as well as NF-kB, cannot stimulate the transcription of genes that are involved in the synthesis of adhesion molecules and pro-inflammatory cytokines such as TNF-α (Orth et al. 1999, Orth, 2002 and Palmer et al., 1999).
YopP seems to be a protease, possibly of the de-SUMOylating family (where SUMO stands for ‘small ubiquitin-related modifier’). The predicted secondary structure of YopP is similar to that of cystein protease from adenovirus (AVP). Mutations in catalytic triad (His109, Glu128 and Cys 172 in YopP) disable YopP to inhibit either the MAPK or the NF-kB pathway.